Without the placenta, the embryo cannot develop. Thus, differentiation of its specialized epithelial cells, termed trophoblasts, precedes that of the embryo proper. In humans, little is known about the initial differentiation process that gives rise to the trophoblast lineage. We have studied the subsequent process whereby cytotrophoblast stem cells present in chorionic villi differentiate. In floating villi, these stem cells detach from their basement membrane and fuse to form an overlying syncytium which is in direct contact with maternal blood. These multinucleate syncytotrophoblasts play an important role in nutrient, waste, and gas exchange. In anchoring villi, cytotrophoblasts also fuse, but many remain as single cells that attach to and invade the uterus and its arterioles. Cytotrophoblast invasion physically connects the embryo/fetus to the uterus, and establishes maternal blood flow to the placenta. Work completed during the previous grant period showed that culturing first trimester human cytotrophoblast stem cells on extracellular matrices (ECMs) redirects their differentiation program along the pathway that leads to invasion, rather than to fusion. During this process, as during uterine invasion, there are dramatic changes in the cells' expression of three classes of molecules. The first class is composed of molecules that are involved in acquisition of an invasive phenotype. Thus, invasive cytotrophoblasts produce and activate the 92 kDa type IV collagenase (MMP-9), and they switch their repertoire of integrin cell-ECM adhesion receptors. In the second class are molecules that are likely to be involved in controlling the maternal immunological response to the invading allogeneic cytotrophoblasts. For example, we showed that class I MHC-null stem cells upregulate their expression HLA- G, a class Ib molecule, when they invade the uterus. The third class of molecules is transcriptional regulators. Data from our laboratory show that the expression of at least one dominant negative regulator of basic helix-loop-helix (bHLH) transcription factors, inhibitors of DNA binding- 2 (Id-2), is downregulated during the differentiation process. This observation suggests that bHLH transcription factors, such as Hxt and Mash-2, which control important aspects of mouse trophoblast differentiation, could regulate similar processes in human cytotrophoblasts. We now propose using our in vitro model of early gestation (8-20 wk) cytotrophoblast differentiation along the invasive pathway to investigate the role of negative (Aim 1) and positive (Aim 2) HLH transcription factors in this process. We will use the resulting information to determine whether similar transcriptional mechanisms could regulate trophoblast differentiation at other critical junctures in the formation of the placenta (Aim 3). Specifically, with regard to the beginning of pregnancy, we will determine whether human embryonic trophoblasts express HLH factors (and stage specific antigens) implicated in the differentiation and invasion of later gestation (8-20 wk) cytotrophoblasts. We will also determine the role of Id and bHLH proteins in the process by which term cytotrophoblasts lose the ability to differentiate at the end of pregnancy. Finally (Aim 4), we will investigate whether the expression of factors that govern normal cytotrophoblast differentiation is disregulated in diseases of pregnancy associated with either abnormally shallow (preeclampsia) or aggressive (placental site trophoblastic tumors, gestational choriocarcinomas) cytotrophoblast invasion. Together, the results of these experiments will enhance our understanding of factors that regulate normal cytotrophoblast differentiation, which is key to the success of pregnancy. In addition, we will gain insight into causes of reproductive disorders that are associated with failures in this process.